Glycopeptide and preparation thereof

ABSTRACT

The stereospecific synthesis of a glycopeptide using a triply orthogonal protection scheme is described, in particular, the synthesis of N-acetylglucosaminyl-β-[1,4]-N-acetylmuramylmonopeptide and derivatives thereof. The glycopeptide is useful for the preparation of GMDP and related compounds having a glucosaminyl-β-[1,4]-N-acetylmuramic acid disaccharide core.

This is the national phase application, under 35 USC 371, forPCT/US01/12630, filed 18 Apr. 2001, which claims the priority of60/197,237, filed 18 Apr. 2000 and, 60/255,829, filed 15 Dec. 2000.

FIELD OF THE INVENTION

The present invention relates to the stereospecific synthesis of aglycopeptide using a triply orthogonal protection scheme, in particular,the synthesis of N-acetylglucosaminyl-β-[1,4]-N-acetylmuramylmonopeptide and derivatives thereof.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 4,395,399 to Ovchinnikov et al. disclosed glycopeptides offormula I

wherein Y′ is a residue of an aminoacid or linear peptide of 2 to 5amino acid residues. These glycopeptides are prepared by couplingunblocked muramyl-containing N-acetylamino-sugars of formula II

with blocked aminoacids or peptides. The disaccharide acid of formula IIis obtained from large-scale fermentation of the bacterium Micrococcuslysodeicticus. The peptide portion is produced by conventional syntheticmethods.

Compounds of formula I (hereinafter referred to as “Ovchinnikovglycopeptides”), particularlyN-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) andN-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-glutamic acid (GMDP-A),are orally-active immunomodulators for use in a number of indications.(see, e.g., Ivanov, V. T., et al., Immunologiya No. 2, 4–5 (1996);Adrianova, I. E., et al., Radiobiologiia 32, 566–70 (1992); Palache, A.M., et al., Vaccine 14, 1327–30 (1996); and Khaitov, R. M., et al.,“Immunotheraphy of Infections,” Ed. Masihi, N., 205–211 (Marcel Dekker,Inc., 1994)). For example, compounds of formula I possess adjuvantactivity. Adjuvants are compounds causing non-specific stimulation ofthe immune system in human beings and animals, resulting in an increasedproduction of antibodies and enhancement of protective reaction of theorganism against infection. Adjuvants are used in medicine for themanufacture of vaccines and sera. In addition, U.S. Pat. No. 5,506,204to R. Aston discloses the use of GMDP and GMDP-A for treatment of septicshock.

The semi-synthetic approach for preparing glycopeptides of formula Idescribed above is utilized because the disaccharide core,N-acetyl-(2-deoxy-2-aminoglucopyranosyl)-β-[1,4]-N-acetylmuramic acid,is one of the most difficult glucopyranosyl-glucopyranose disaccharidesto synthesize. For example, the order of glucopyranose hydroxyl acceptorreactivity toward glycosyl cation donors, independent of donor source,is water>>ethanol>C(6)OH>C(2)OH>C(3)OH> the required C(4)OH. Inaddition, 2-deoxy-2-acylaminoglucopyranose C(4)OH acceptors aredeactivated electronically relative to glucose itself. Muramic acidderivatives, in particular, suffer still further acceptor reactivitydisadvantage due to steric crowding around the C(4) oxygen. Formation ofthe desired β-[1,4]-glycosidic bond requires a2-deoxy-2-aminoglucopyranose glycosyl cation donor with a nitrogensubstituent that will favor equatorial approach of the very modestlynucleophilic C(4)OH of a muramic acid derivative.

Several approaches to this formidable glycosidation problem have beendocumented. (see, e.g., Mercer, C., et al., Tetrahedron Lett. 13, 1029(1973); Durette, P. L., et al., Carbohydr. Res., 77, C1 (1979);Kusumoto, D., et al., Bull. Chem. Soc. Jpn., 59, 1411 (1986); Kusumoto,D., et al., Bull. Chem. Soc. Jpn., 59, 1419 (1986); Farkas, J., et al.,Carbohydr. Res., 163, 63 (1987); Kinzy, W., et al., Liebigs Ann. Chem.,407 (1987) ; Termin, A., et al., Liebigs Ann. Chem., 789 (1989);Ledvina, M., et al., Collect. Czech. Chem. Commun., 54, 2784 (1989) andTermin, A., et al., Liebigs Ann. Chem., 527 (1992). However, none ofthese approaches provides a process for preparing the disaccharide insufficient amounts to be useful as an intermediate in the preparation ofglycopeptides of formula I.

SUMMARY OF THE INVENTION

The present invention provides a process for preparing a protectedglycopeptide of formula 1

by coupling a muramylamide compound of formula 2

with a glucopyranosyl compound of formula 3

to form the protected glycopeptide of formula 1, wherein:

A is Br or Cl;

Pg⁰ is an acyl hydroxy-protecting group;

Pg¹ is a hydroxy-protecting group which is not electron withdrawing;

Pg² is an amine-protecting group which does not lead to oxazolineformation;

Pg⁵ is a hydroxy-protecting group;

Pg⁰, Pg¹, Pg², and Pg⁵ are mutually orthogonal protecting groups; and

Y is a residue of an amino acid or peptide, wherein:

-   -   Y forms an amide linkage with the attached carbonyl; and    -   Y comprises a protected terminal carboxy group.

The invention also provides compounds of formula III

wherein:

R⁰ is Pg⁰ or hydrogen;

R¹ is Pg¹, Pg³ or hydrogen;

R² is Pg² or acetyl;

R^(Y) is Y or Y′;

Pg⁰ is an acyl hydroxy-protecting group;

Pg¹ is a hydroxy-protecting group which is not electron-withdrawing;

Pg² is a an amine-protecting group which does not lead to oxazolineformation;

Pg³ is an acyl hydroxy-protecting group;

Pg⁵ is a hydroxy-protecting group;

Pg⁰, Pg¹, Pg² and Pg⁵ are mutually orthogonal protecting groups;

Y′ is a residue of an amino acid or peptide, wherein:

-   -   Y′ forms an amide linkage with the attached carbonyl; and    -   Y comprises an unprotected terminal carboxy group;

Y is a residue of an amino acid or peptide, wherein:

-   -   Y forms an amide linkage with the attached carbonyl; and    -   Y comprises a protected terminal carboxy group.        Compounds of formula III are useful intermediates in the        synthesis of the Ovchinnikov glycopeptides.

DETAILED DESCRIPTION Definitions

As used above, and throughout the description of the invention, thefollowing abbreviations, unless otherwise indicated, shall be understoodto have the following abbreviations:

Designation Reagent or Fragment Ac —C(O)CH₃ AcOH acetic acid Ac₂O aceticanhydride BOC t-butyloxycarbonyl DBU 1,8-diazabicyclol[5.4.0]undec-7-eneTHF tetrahydrofuran TsOH p-toluenesulfonic acid NMM N-methylmorpholineTFA trifluoroacetic acid Troc 2,2,2-trichioroethoxycarbonyl min minutesh hour(s) Cbz benzyloxycarbonyl TLC thin layer chromatography NMRnuclear magnetic resonance ESI-MS electro-spray ionization mass spectumEtOAc ethyl acetate IR infrared spectrum MeOH methanol NaOMe sodiummethoxide NHS N-hydroxysuccinimide EDCI 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride

As used above, and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

“Amino acid” means an amino acid selected from the group consisting ofnatural and unnatural amino acids as defined herein. Amino acid is alsomeant to include-amino acids having L or D stereochemistry at theα-carbon. Preferred amino acids are those possessing an α-amino group.The amino acids may be neutral, positive or negative depending on thesubstituents in the side chain. “Neutral amino acid” means an amino acidcontaining uncharged side chain substituents. Exemplary neutral aminoacids include alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, methionine, glycine, serine, threonine andcysteine. “Positive amino acid” means an amino acid in which the sidechain substituents are positively charged at physiological pH. Exemplarypositive amino acids include lysine, arginine and histidine. “Negativeamino acid” means an amino acid in which the side chain substituentsbear a net negative charge at physiological pH. Exemplary negative aminoacids include aspartic acid and glutamic acid. Preferred amino acids areα-amino acids. Exemplary natural amino acids are isoleucine, proline,phenylalanine, tryptophan, methionine, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine,aspartic acid and glutamic acid. Unnatural amino acid” means an aminoacid for which there is no nucleic acid codon. Examples of unnaturalamino acids include, for example, the D-isomers of the natural α-aminoacids as indicated above; Aib (aminobutyric acid), βAib(3-amino-isobutyric acid), Nva (norvaline), βAla, Aad (2-aminoadipicacid), βAad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba(γ-aminobutyric acid), Acp (6-aminocaproic acid), Dbu(2,4-diaminobutryic acid), α-aminopimelic acid, TMSA(trimethylsilyl-Ala), aIle (allo-isoleucine), Nle (norleucine),tert-Leu, Cit (citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr(2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala),hydroxyproline, Sar (sarcosine), and the like; cyclic amino acids;N^(a)-alkylated amino acids such as MeGly (N^(a)-methylglycine), EtGly(N^(a)-ethylglycine) and EtAsn (N^(a)-ethylasparagine); and amino acidsin which the α-carbon bears two side-chain substituents. The names ofnatural and unnatural amino acids and residues thereof used hereinfollow the naming conventions suggested by the IUPAC Commission on theNomenclature of Organic Chemistry and the IUPAC-IUB Commission onBiochemical Nomenclature as set out in “Nomenclature of a-Amino Acids(Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent thatthe names and abbreviations of amino acids and residues thereof employedin this specification and appended claims differ from those noted,differing names and abbreviations will be made clear.

“Amino acid protecting group” and “peptide-protecting group” mean agroup that protects an acid or amine moiety of the amino acid/peptide orother reactive moiety on the side chain of an amino acid/amino acidresidue, e.g., hydroxy or thiol. For examples of “correspondingprotected derivatives” of amino acid side chains, see T. W. Green and P.G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley andSons, 1991. Protecting groups for an acid group in an amino acid aredescribed herein in the section “carboxy-protecting group.” Protectinggroups for an amine group in an amino acid are described in the section“amine-protecting group.”

“Amino acid residue” means the individual amino acid units incorporatedinto a peptide, or peptide portion of a molecule, through an amidelinkage.

“Amine-protecting group” means an easily removable group that is knownin the art to protect an amino group against undesirable reaction duringsynthetic procedures and to be selectively removable. The use ofamine-protecting groups is well known in the art for protecting groupsagainst undesirable reactions during a synthetic procedure and many suchprotecting groups are known, for example, T. H. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley &Sons, New York (1991), incorporated herein by reference. Amineprotecting group also includes acid-labile amine-protecting groups(e.g., BOC) and hydrogenation-labile amine-protecting groups (e.g.,Cbz). In the present invention, Pg² is a group which does not lead tothe generation of undesirable oxazoline by-products (i.e., Pg² cannot bean acyl group). Suitable amine-protecting groups include carbamate andimide groups. Particular imide groups include phthalimide,tetrachlorophthalimide and (Ac)₂N—. Particular carbamate groups includemethoxy-carbonyl, 9-fluorenylmethoxycarbonyl,2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl,vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC),1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (CBZ),p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl,trimethylsilyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,1,1-dimethyl-2,2,2-trichloroethoxycarbonyl, and the like. A preferredamine-protecting group is 2,2,2-trichloroethoxycarbonyl.

“Carboxy-protecting group” means an easily removable group that is knownin the art to protect an acidic hydrogen of a carboxyl group againstundesirable reaction during synthetic procedures, e.g., to block orprotect the acid functionality while the reactions involving otherfunctional sites of the compound are carried out, and to be selectivelyremovable. Such acid protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups, as described in U.S. Pat. Nos. 3,840,556 and 3,719,667, thedisclosures of which are hereby incorporated herein by reference. Forsuitable acid protecting groups, see T. W. Green and P. G. M. Wuts in“Protective Groups in Organic Chemistry” John Wiley and Sons, 1991. Acidprotecting group also includes hydrogenation labile acid protectinggroups, such as benzyl. Examples of acid protecting groups includeesters such as substituted and unsubstituted C₁ to C₈ alkyl, e.g.,methyl, ethyl, t-butyl, methoxymethyl, methylthiomethyl,2,2,2-trichloroethyl and the like, tetrahydropyranyl, substituted andunsubstituted phenylalkyl such as benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like,cinnamyl, dialkylaminoalkyl, e.g., dimethylaminoethyl and the like,trimethylsilyl, substituted and unsubstituted amides and hydrazides,e.g., amides and hydrazides of N,N-dimethylamine, 7-nitroindole,hydrazine, N-phenylhydrazine and the like, acyloxyalkyl groups such aspivaloyloxymethyl or propionyloxymethyl and the like, aroyloxyalkyl suchas benzoyloxyethyl and the like, alkoxycarbonylalkyl such asmethoxycarbonylmethyl, cyclohexyloxycarbonylmethyl and the like,alkoxycarbonyloxyalkyl such as t-butyloxycarbonyloxymethyl and the like,alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike, alkylaminocarbonylaminoalkyl, such asmethylaminocarbonylaminomethyl and the like, acylaminoalkyl such asacetylaminomethyl and the like, heterocyclylcarbonyloxyalkyl such as4-methylpiperazinyl-carbonyloxymethyl and the like,dialkylaminocarbonylalkyl such as dimethylaminocarbonyl-methyl and thelike, (5-(lower alkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like, and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like. Particularcarboxy-protecting groups include methyl, 9-fluorenylmethyl,2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl,2-methylthioethyl, 1,3-dithianyl-2-methyl, 2-(p-toluenesulfonyl)ethyl,2-(p-nitrophenylsulfenyl)ethyl. 2-(2′-pyridyl)ethyl,2-(diphenylphosphino)ethyl, p-(methylmercapto)phenyl, nitroethyl, allyland the like. Preferred carboxy-protecting groups are cyanoethyl,t-butyl and —CH₂CH₂SO₂Ph.

“Hydroxy-protecting group” means an easily removable group that is knownin the art to protect an hydroxyl group against undesirable reactionduring synthetic procedures and to be selectively removable. The use ofhydroxy-protecting groups is well known in the art for protecting groupsagainst undesirable reactions during a synthetic procedure and many suchprotecting groups are known, for example, T. H. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley &Sons, New York (1991), incorporated herein by reference. In the presentinvention, the Pg⁰, Pg¹, and Pg⁵ hydroxy-protecting groups are mutuallyorthogonal, as described herein. Pg¹ cannot be an electron-withdrawinggroup, since such groups deactivate the coupling reaction between themuramylamide compound of formula 2 and glucosopyranosyl compound offormula 3. Suitable Pg¹ groups include aralkyl, aralkenly and silylgroups. Particular aralkyl and alkenyl groups include benzyl and allyl,respectively. Particular silyl groups include trialkylsilyl groups, suchas trimethylsilyl and (t-butyl)dimethylsilyl. Preferred Pg¹ groups areallyl and benzyl; a more preferred group is benzyl. In addition, Pg⁰ andPg³ must be removable by saponification (i.e., Pg⁰ and Pg³ must be acylgroups). Particular acyl groups include formyl, acetyl, chloroacetyl,trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl,trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl,o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl,and the like. Preferred groups are chloroacetyl and acetyl; a morepreferred group is acetyl. Finally, since Pg⁵ is orthogonal to the otherhydroxy-protecting groups, it must be stable under saponificationconditions (i.e., Pg⁵ cannot an acyl group) and some conditions suitablefor removal of Pg¹. Suitable Pg⁵ groups aralkyl and alkenyl groups.Preferred Pg⁵ groups include allyl, n-pentenyl, and benzyl; a morepreferred group is benzyl.

“Leaving group” of an activated ester means a substituent havingsufficient lability such that it can be substituted by a goodnucleophile (i.e., an amino group of a peptide unit). The lability of aparticular substituent will vary depending upon substituents on the sameand/or adjacent carbon atoms and the nature of the leaving group. Thoseskilled in the art will appreciate the types of leaving groups capableof substitution by an amino nucleophile. For suitable leaving groups,see M. Bodanszky and A. Bodanszky in “The Practice of Peptide Synthesis”Springer-Verlag, 1984; and M. Bodanszky in “Principles of PeptideSynthesis” Springer-Verlag, 1984. In the present invention, the leavinggroup activates the attached carbonyl such that the terminal amino acidgroup acts as a linker for linking the disaccharide with the peptideunit. Particular leaving groups include pentafluorophenoxy,N-oxysuccimide, N-oxyphthalimide, and N-oxybenzotriazole. A preferredleaving group is N-oxysuccinimide.

“Orthogonal protecting groups” means protecting groups for which thereexists a set of conditions wherein one of the groups can be removedwithout removing the other(s). The term encompasses protecting groupsfor different moieties (e.g., orthogonal amine and hydroxy protectinggroups) as well as the same moiety (e.g., orthogonal hydroxy-protectinggroups). It is not a requirement that orthogonal protecting groupsnecessarily be different. For example, when the term is used to describeprotecting groups for the same moiety, the groups may be different(e.g., orthogonal acetyl and benzyl hydroxy-protecting groups) or thesame (e.g., orthogonal benzyl protecting groups). “Electron-withdrawinggroup” means a group which is a more powerful electron attractor thanhydrogen. Electron withdrawing groups exhibit negative inductiveeffects, whereas groups which are poorer electron attractors thanhydrogen exhibit positive inductive effects. (see, e.g., E. S. Gould,Mechanism and Structure in Organic Chemistry, Holt, Rinehart andWinston, New York (1959), incorporated herein by reference).

“Acyl” means an R—C(O)— group, wherein R is bonded to the CO groupthrough a carbon-carbon bond.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched having about 1 to about 20 carbon atoms in the chain. Preferredalkyl groups have 1 to about 12 carbon atoms in the chain, morepreferred is lower alkyl as defined herein. Branched means that one ormore lower alkyl groups such as methyl, ethyl or propyl are attached toa linear alkyl chain. “Lower alkyl” means about 1 to about 4 carbonatoms in the chain that may be straight or branched.

“Alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched havingabout 2 to about 15 carbon atoms in the chain. Preferred alkenyl groupshave 2 to about 12 carbon atoms in the chain; and more preferably about2 to about 4 carbon atoms in the chain. Branched means that one or morelower alkyl groups such as methyl, ethyl or propyl are attached to alinear alkenyl chain. “Lower alkenyl” means about 2 to about 4 carbonatoms in the chain that may be straight or branched. Exemplary alkenylgroups include ethenyl, propenyl, n-butenyl, i-butenyl,3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl anddecenyl.

“Aryl” means an aromatic monocyclic or multicyclic ring system of about6 to about 14 carbon atoms, preferably of about 6 to about 10 carbonatoms. Exemplary aryl groups include phenyl or naphthyl, or phenylsubstituted or naphthyl substituted.

“Carboxy” means an HO(O)C— (carboxylic acid) group.

“N-oxysuccinimide” means a moiety of the following

structure

“Peptide” means a polymer encompassing amino acid residues joinedtogether through amide bonds.

“GMDP” refers toN-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine, which hasthe following structure:

“GMDP-A” refers to theN-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-glutamic acid, which hasthe following structure:

With reference to formulas 1–19, as described herein, particular andpreferred embodiments are as follows:

In a first particular embodiment of the invention, the muramylamidecompound of formula 2, as described herein, and glucopyranosyl compoundof formula 3, as described herein, are reacted under scrupulouslyanhydrous conditions.

In a second particular embodiment of the invention, Pg⁰ is acetyl.

In a third particular embodiment of the invention, the Pg⁰ group of acompound of formula 8, as described herein, is removed to form acompound of formula 7, as described herein.

In a fourth particular embodiment of the invention, the Pg⁰ group of acompound of formula 13, as described herein, is removed to form acompound of formula 12, as described herein.

In a preferred embodiment, the Pg⁰ group is removed in the presence ofaqueous sodium hydroxide.

In a fifth particular embodiment of the invention, Pg⁵ is benzyl, allylor n-pentenyl.

In a preferred embodiment, Pg⁵ is benzyl.

In a sixth particular embodiment of the invention, the Pg⁵ group of acompound of formula 12, as described herein, is removed to form acompound of formula 11, as described herein.

In a seventh particular embodiment of the invention, the Pg⁵ group of acompound of formula 7, as described herein, is removed to form acompound of formula I, as described herein.

In a preferred embodiment, the Pg⁵ group is removed in the presence ofhydrogen and a palladium/carbon catalyst.

In an eighth particular embodiment of the invention, Pg¹ is a benzyl,allyl or silyl hydroxy-protecting group;

In a preferred embodiment, Pg¹ is benzyl.

In a preferred embodiment, a muramylamide compound of formula 3a, asdescribed herein, is prepared by reductively opening the 1,3-dioxanering of a muramylamide of formula 6, as described herein.

In a ninth particular embodiment of the invention, the Pg¹ group of acompound of formula 1, as described herein, is exchanged with a Pg³group to form a compound of formula 10, as described herein.

In a tenth particular embodiment of the invention, the Pg¹ group of acompound of formula 1a, as described herein, is exchanged with a Pg³group to form a compound of formula 19, as described herein.

In a preferred embodiment, Pg³ is acetyl.

In a more preferred embodiment, the exchanging is carried out in thepresence of acetic anhydride, acetic acid, and zinc chloride.

In a eleventh particular embodiment of the invention, Pg² is a carbamateor imide amine-protecting group;

In a preferred embodiment, Pg² is 2,2,2-trichloroethoxycarbonyl.

In an twelfth particular embodiment of the invention, the Pg² group of acompound of formula 19, as described herein, is exchanged with an acetylgroup to form a compound of formula 18, as described herein.

In a thirteenth particular embodiment of the invention, the Pg² group ofa compound of formula 10, as described herein, is exchanged with anacetyl group to form a compound of formula 9, as described herein.

In a preferred embodiment, the exchanging is carried out in the presenceof acetic anhydride, acetic acid, and zinc dust.

In a fourteenth particular embodiment of the invention, the Pg³ group ofa compound of formula 8, as described herein, is removed to form acompound of formula 7, as described herein.

In a fifteenth particular embodiment of the invention, the Pg³ group ofa compound of formula 13, as described herein, is removed to form acompound of formula 12, as described herein.

In a preferred embodiment, the Pg³ group is removed in the presence ofaqueous sodium hydroxide.

In a sixteenth particular embodiment of the invention, LOH isN-hydroxysuccinimide.

In a seventeenth particular embodiment of the invention, A is Br.

In an eighteenth particular embodiment of the invention, Y is a peptidecomprising 2 to 5 amino acid residues.

In a preferred embodiment, Y is a linear peptide.

In an nineteenth particular embodiment of the invention, each of X′ andW′ is a residue of an amino acid or peptide comprising 2 to 4 amino acidresidues, provided that the total number of amino acid residues in X′and W′ is 2 to 5;

In a preferred embodiment, each of X′ and W′ is a linear peptide.

In a more preferred-embodiment, the —X′—W′ is a linear peptide;

With reference to formula III above, particular and preferredembodiments are as follows:

In a twentieth particular embodiment of the invention, R⁰ is Pg⁰.

In a preferred embodiment, Pg⁰ is acetyl.

In a twenty-first particular embodiment of the invention, R⁰ ishydrogen.

In a twenty-second particular embodiment of the invention, R¹ is Pg¹.

In a preferred embodiment, Pg¹ is benzyl, allyl or silyl.

In a more preferred embodiment, Pg¹ is benzyl.

In a twenty-third particular embodiment of the invention, R¹ is Pg³.

In a preferred embodiment, Pg³ is acetyl.

In a twenty-fourth particular embodiment of the invention, R¹ ishydrogen.

In a twenty-fifth particular embodiment of the invention, R² is Pg².

In a preferred embodiment, Pg² a carbamate or imide amine-protectinggroup;

In more preferred embodiment, Pg² is 2,2,2-trichloroethoxycarbonyl.

In a twenty-sixth particular embodiment of the invention, R² is acetyl.

In a twenty-seventh particular embodiment of the invention, Pg⁵ isbenzyl, allyl or n-pentenyl.

In a preferred embodiment, Pg⁵ is benzyl.

In a twenty-eighth particular embodiment of the invention, Y is apeptide comprising 2 to 5 amino acid residues.

In a preferred embodiment, Y′ is a linear peptide.

In a twenty-ninth particular embodiment of the invention, Y′ is apeptide comprising 2 to 5 amino acid residues.

In a preferred embodiment, Y′ is a linear peptide.

This invention also includes all combinations of particular andpreferred embodiments described herein.

Preparation of Compounds of Formula 1

Synthesis of a compound of formula 1, the central disaccharide core, inorthogonally protected form presents a significant synthetic challenge.For example, identification of a protective scheme having tripleorthogonality is highly desirable to accomplish selective unmasking ofthe three types of pendant hydroxyl groups (i.e., anomeric OH,peripheral OH, and carboxyl OH). In addition, the stereoselectiveconstruction of the β-[1,4] glycosidic linkage is expected to bedifficult irrespective of the method used to generate a reactiveglycosyl cation donor. For example, with respect to the glycosyl cation,each of the following inherent properties contribute to a loss ofreactivity of the glycosyl cation acceptor: (i) the intrinsic lack ofnucleophilicity of the C(4)-hydroxyl group of glucopyranose-basedacceptors, (ii) additional steric crowding around the C(4)-hydroxyl-ofmuramic acid-based acceptors, and (iii) additional electronicdeactivation of 2-deoxy-2-acylaminoglucopyranose acceptors relative totheir glucopyranose-based counterparts. With respect to the glycosylcation donor, an activation method with a predisposition towardformation of a β-[1,4] glycosidic linkage is needed. The reactionconditions for glycosyl cation generation also needs to be compatiblewith functionality resident in both the donor and acceptor.

A compound of formula 1, wherein the variables are as described herein,may be prepared by coupling a muramylamide compound of formula 2,wherein the variables are as

described herein, with a glucopyranosyl compound of formula 3, whereinthe variables are as described herein, under appropriate conditions.Particular conditions include carrying out the coupling reaction underscrupulously anhydrous Konigs-Knorr conditions (e.g., in a silvertriflate/dichloromethane solution including molecular sieves), or thelike.

A compound of formula 2 is prepared according to the proceduresdescribed in Imoto, M., Bull. Chem. Soc. Jpn., 60, 2205 (1987).

A compound of formula 3, wherein the variables are as described herein,may be prepared by coupling an acid of formula 4, wherein the variablesare as described herein,

with a protected amino acid/peptide compound of formula 5, wherein Y isas described herein, under appropriate conditions. Particular conditionsencompass carrying out the coupling reaction in a solution of NMM (orthe like) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (or the like) inCH₂Cl₂ (or the like), wherein the compound of formula 5 is added as thetosylate salt, or the like.

A compound of formula 3a, wherein Pg¹ is benzyl and the other variablesare as described herein, may be prepared by treating a compound offormula 6, wherein the variables are

as defined herein, with a reducing agent under appropriate conditions. Aparticular reducing agent is triethylsilane, or the like. Particularconditions include carrying out the reduction in CH₂Cl₂ (or the like)and TFA (or the like) at about 0° C. This reaction provides an efficientmeans for regioselective introduction of the benzylprotection/activation at C(6)OH of the muramic acid derivative.

It is known that when an ester derivative of a compound of formula 6 istreated with trifluoroacetic acid and triethylsilane as described inDeNinno, M. P., et al., Tetrahedron Lett., 36, 669 (1995), only a smallamount of the analogous compound of formula 3a is observed. The majorproduct formed is a lactone, as shown in scheme I.

The acid-catalyzed lactonization proceeds at a rate competitive with thereductive ring opening, thus leading to the undesired lactone. In thepresent invention, however, introduction of an amide bond in place ofthe ester bond eliminates the conversion to the lactone, thus allowingthe desired product (compound 3a) to be isolated in much higher yields.

A compound of formula 6, wherein the variables are as described herein,may be prepared by coupling an acid of formula 4a, wherein the variablesare as described herein,

with a protected amino acid/peptide compound of formula 5, wherein Y isas described herein, under appropriate conditions. Particular conditionsencompass carrying out the coupling reaction in a solution of NMM (orthe like) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (or the like) inCH₂Cl₂ (or the like) , wherein the compound of formula 5 is added as thetosylate salt, or the like.

The synthesis of the core disaccharide outlined above provides a highthroughput access to intermediates in the total synthesis of theOvchinnikov glycopeptides. Due to the convergent approach, protectinggroup economy, and crystalline nature of the intermediates, one caneasily scale the process to substantial or commercial volumes.

Preparation of Compounds of Formula I

I. Direct Attachment of the Amino Acid/Peptide Portion

A Compounds of formula I, wherein Y′ is the unprotected form of Y, maybe prepared by removing the Pg⁵ group of a compound of formula 7,wherein the variables are as described herein, in the presence of ahydroxy-deprotecting agent and under appropriate conditions.

A particular Pg⁵ group is benzyl, or the like. A particularhydroxy-deprotecting agent is H₂/(Pd/C), or the like. Particularhydroxy-deprotecting conditions encompass carrying out the deprotectionin an alcohol solvent (e.g., methanol, ethanol or the like) at aboutroom temperature.

A compound of formula 7, wherein the variables are as described herein,may be prepared by saponifying the Pg⁰ and Pg³ groups of a compound offormula 8,

wherein Pg³ is an acyl hydroxy-protecting group and the other variablesare as described herein, in the presence of a saponifying agent andunder appropriate conditions. A particular Pg⁰ and Pg³ group is acetyl,or the like. A particular saponifying agent is aqueous sodium hydroxide,or the like. Particular saponifying conditions encompass carrying outthe saponification in an alcohol solvent (e.g., methanol, ethanol or thelike) at about room temperature.

A compound of formula 8, wherein the variables are as defined herein,may be prepared by deprotecting the Y group a compound of formula 9,wherein the variables are as

described herein, under appropriate conditions. The peptide-deprotectingis carried out using an appropriate deprotecting agent that depends onthe nature of the peptide-protecting group, i.e., whether it isremovable (labile) under acid, base, or hydrogenation conditions, andother reactive moieties in the compound undergoing deprotection, i.e., adeprotecting agent is chosen to carry out the deprotection withoutaffecting the other reactive moieties unless a concomitant reaction isdesired. A particular peptide-protecting group for a carboxylic acidmoiety is C₁ to C₈ alkyl; more particularly t-butyl, or the like. Aparticular peptide-deprotecting agent for such a group is an inorganicacid; more particularly HCl, or the like. Another particularpeptide-protecting group for a carboxylic acid moiety is —CH₂CH₂SO₂Ph,or the like. A particular peptide-deprotecting agent for such a group isDBU (or the like), wherein particular conditions encompass dissolvingthe compound of formula 9 in THF (or the like) and adding DBU (or thelike) dropwise as a THF solution (or the like).

A compound of formula 9, wherein the variables are as described herein,may be prepared by exchanging the Pg² group of compound of formula 10,wherein the variables are as described herein, with an acetyl group inthe presence of

an amine-deprotecting agent and acylating agent under appropriateconditions. The amine-deprotecting is carried out using an appropriatedeprotecting agent that depends on the nature of the amine-protectinggroup, i.e., whether it is removable (labile) under acid, base, orhydrogenation conditions, and other reactive moieties in the compoundundergoing deprotection, i.e., a deprotecting agent is chosen to carryout the deprotection without affecting the other reactive moietiesunless a concomitant reaction is desired. A particular amine-protectinggroup is β,β,β-trichloroethoxycarbonyl, or the like. A particulardeprotecting agent is Zn dust (or the like) in the presence of a protonsource (e.g., acetic acid or the like). A particular acylating agent isacetic anhydride, or the like. Particular conditions include adding Zndust (or the like) and about a 3:2:1 mixture of THF:Ac₂O:AcOH (or thelike) to a solution of the compound of formula 10 in about a 2:1 mixtureof Ac₂O:AcOH, or the like.

A compound of formula 10, wherein the variables are as described herein,may be prepared by exchanging the Pg¹ group a compound of formula 1,wherein the other variables are as described herein, with a Pg³ group inthe presence

of a solvolyzing agent and acylating agent under appropriate conditions.The solvolysis is carried out using an appropriate solvolyzing agentthat depends on the nature of the hydroxy-protecting group, i.e., asolvolyzing agent is chosen to carry out the solvolysis withoutaffecting the other reactive moieties unless a concomitant reaction isdesired. A particular hydroxy-protecting group is benzyl, or the like. Aparticular solvolyzing agent is ZnCl₂, or the like. A particularacylating agent is acetic anhydride, or the like. Particular conditionsinclude carrying out the solvolysis/acylation in about a 2:1 mixture ofAc₂O:AcOH, or the like.II. Attachment of the Amino Acid/Peptide Portion in Stages

A compound of formula I, wherein —Y′ is —X′—W′, X′ is a residue of anamino acid or peptide which forms an amide linkage with the attachedcarbonyl, and W′ is a residue of an amino acid or peptide, may beprepared by deprotecting the W group of a compound of formula 11 in thepresence of a

peptide-deprotecting agent under appropriate conditions. Thepeptide-deprotecting is carried out using an appropriate deprotectingagent that depends on the nature of the peptide-protecting group, i.e.,whether it is removable (labile) under acid, base, or hydrogenationconditions, and other reactive moieties in the compound undergoingdeprotection, i.e., a deprotecting agent is chosen to carry out thedeprotection without affecting the other reactive moieties unless aconcomitant reaction is desired. A particular peptide-protecting groupfor a carboxylic acid moiety is C₁ to C₈ alkyl; more particularlyt-butyl, or the like. A particular peptide-deprotecting agent for such agroup is an inorganic acid; more particularly HCl, or the like. Anotherparticular peptide-protecting group for a carboxylic acid moiety is—CH₂CH₂SO₂Ph, or the like. A particular peptide-deprotecting agent forsuch a group is DBU (or the like), wherein particular conditionsencompass dissolving the compound of formula 11 in THF (or the like) andadding DBU (or the like) dropwise as a THF solution, or the like.

A compounds of formula 11 may be prepared by removing the Pg⁵ group of acompound of formula 12, wherein the variables are as described herein,in the-presence of a hydroxy-deprotecting agent and under appropriateconditions.

A particular protecting group is benzyl, or the like. A particularhydroxy-deprotecting agent is H₂/(Pd/C carbon), or the like. Particularhydroxy-deprotecting conditions encompass carrying out the deprotectionin an alcohol solvent (e.g., methanol, ethanol or the like) at aboutroom temperature.

A compound of formula 12, wherein the variables are as described herein,may be prepared by saponifying the Pg⁰ and Pg³ groups of a compound offormula 13,

wherein the variables are as described herein, in the presence of asaponifying agent under appropriate conditions. A particular saponifyingagent aqueous sodium hydroxide, or the like. Particular conditionsencompass carrying out the saponification in an alcohol solvent (e.g.,methanol, ethanol or the like) at about room temperature.

A compound of formula 13, wherein the variables are as described herein,may be prepared by coupling a compound of formula 15, wherein —X″C(O)OLis the activated ester of —X′, —OL is a leaving group capable ofsubstitution by an amino nucleophile, and the other variables area asdescribed herein, with a protected amino acid/peptide of formula 14,wherein W is as defined herein, and under appropriate conditions.

A particular protected amino acid is γ-Obu^(t)-iso-Gln, or the like.Particular conditions encompass adding dropwise a solution ofγ-Obu^(t)-iso-Gln or the like (in about a 2:1 mixture ofacetonitrile:DMF, or the like) to a solution of compound 15 (inacetonitrile or the like), followed immediately by diisopropylethylamine, or the like.

A compound of formula 15, wherein the variables are as described herein,may be prepared by esterifying an acid of formula 17, wherein thevariables are as defined herein, with a compound of formula 16, whereinthe variables are as described herein, under appropriate conditions.

A particular compound of formula 16 is N-hydroxysuccinimide, or thelike. Particular conditions encompass forming a slurry of the compoundof formula 17 in acetonitrile (or the like), and adding EDCI (or thelike) and N-hydroxysuccinimnide (or the like) to the slurry at aboutroom temperature.

A compound of formula 17, wherein the variables are as described herein,may be prepared by deprotecting the terminal carboxy moiety of the Xgroup of a compound of formula 18, wherein the variables are asdescribed herein, with an appropriate amino acid/peptide deprotectingagent

under appropriate conditions. The peptide-deprotecting is carried outusing an appropriate deprotecting agent that depends on the nature ofthe carboxy-protecting group, i.e., whether it is removable (labile)under acid, base, or hydrogenation conditions, and other reactivemoieties in the compound undergoing deprotection, i.e., a deprotectingagent is chosen to carry out the deprotection without affecting theother reactive moieties unless a concomitant reaction is desired. Aparticular peptide-protecting group for a carboxylic acid moiety is C₁to C₈ alkyl; more particularly t-butyl, or the like. A particularpeptide-deprotecting agent for such a group is an inorganic acid; moreparticularly HCl, or the like. Another particular peptide-protectinggroup for a carboxylic acid moiety is —CH₂CH₂SO₂Ph, or the like. Aparticular peptide-deprotecting agent for such a group is DBU (or thelike), wherein particular conditions encompass dissolving the compoundof formula 18 in THF (or the like) and adding DBU (or the like) dropwiseas a THF solution, or the like.

A compound of formula 18, wherein the variables are as described herein,may be prepared by exchanging the Pg² group of a compound of formula 19,wherein the variables are as described herein, with an acetyl group inthe presence of

an amine-deprotecting agent and an acylating agent under the appropriateconditions. The amine-deprotecting is carried out using an appropriatedeprotecting agent that depends on the nature of the amine-protectinggroup, i.e., whether it is removable (labile) under acid, base, orhydrogenation conditions, and other reactive moieties in the compoundundergoing deprotection, i.e., a deprotecting agent is chosen to carryout the deprotection without affecting the other reactive moietiesunless a concomitant reaction is desired. A particular amine-protectinggroup is β,β,β-trichloroethoxycarbonyl, or the like. A particulardeprotecting agent is Zn dust (or the like) in the presence of a protonsource (e.g., acetic acid or the like). A particular acylating agent isacetic anhydride, or the like. Particular conditions include adding Zndust (or the like) and about a 3:2:1 mixture of THF:Ac₂O:AcOH (or thelike) to a solution of the compound of formula 8 in about a 2:1 mixtureof Ac₂O:AcOH, or the like.

A compound of formula 19, wherein the variables are as described herein,may be prepared by exchanging the Pg¹ group of a compound of formula 1a,wherein the variables are as described herein, with a Pg³ group in thepresence of a

solvolyzing agent and acylating agent under appropriate conditions. Thesolvolysis is carried out using an appropriate solvolyzing agent thatdepends on the nature of the hydroxy-protecting group, i.e., asolvolyzing agent is chosen to carry out the solvolysis withoutaffecting the other reactive moieties unless a concomitant reaction isdesired. A particular hydroxy-protecting group is benzyl, or the like. Aparticular solvolyzing agent is ZnCl₂, or the like. A particularacylating agent is acetic anhydride, or the like. Particular conditionsinclude carrying out the solvolysis/acylation in about a 2:1 mixture ofAc₂O:AcOH, or the like.

It is understood that the process described above can be modified sothat the peptide portion can attached in three or more stages.

EXAMPLES General

Reactions are carried out with continuous stirring under a positivepressure of nitrogen except where noted. Dilutions/solutions of liquidsare shown as volume:volume. Reagents and solvents are purchased and usedwithout further purification. TLC is performed with 0.25 mm silica gel60 plates with a 254 nm fluorescent indicator from E. Merck. Plates aredeveloped in a covered chamber and visualized by ultraviolet light or bytreatment with 5% phosphomolybdic acid in ethanol followed by heating.Flash chromatography is carried out with silica gel 60, 230–400 mesh(0.040–0.063 mm particle size) purchased from EM Science. HPLC analysesand purifications are performed using Dynamax C8 columns with thespecified solvent system and flow rate. NMR spectra are reported aschemical shifts in parts-per-million (ppm) downfield from atetramethylsilane internal standard (0 ppm). ¹H NMR spectra are recordedin the solvent indicated on either a Bruker Avance spectrometer at500.18 MHz, a Varian Mercury spectrometer at 400.21 MHz, or a GE QE-300spectrometer at 300.15 MHz. ¹³C NMR spectra are recorded in the solventsindicated on the previously mentioned spectrometers at 125.78 MHz,100.15 MHz, and 75.48 MHz, respectively. IR spectra are recorded on aNicolet 510P FT-IR spectrometer; electrospray mass spectra are recordedon a Micromass Platform LCZ spectrometer. High resolution mass spectraare recorded on a Micromass QTOF mass spectrometer.

The synthetic process for preparation of a protected disaccharide,compound vi, is outlined in scheme II and exemplified in Example 1, bothshown below.

Example 1

I. Regioselective Installation of Benzyl Protection & Attachment ofPeptide Linker:

A mixture of (L)-alanine (15.0 g, 168 mmol), phenylsulfonyl ethanol(37.6 g, 202 mmol), and TsOH.H₂O (35.2 g, 185 mmol) in benzene (750 mL)is refluxed using a Dean-Stark apparatus. After 16 h, additionalphenylsulfonyl ethanol (25 g, 135 mmol) and TsOH.H₂O (25 g, 134 mmol) isadded along with benzene (180 mL), and the reaction mixture is refluxedovernight. Concentration in vacuo gives the product, compound i, inquantitative yield as a white solid.

Analytical (compound i): ¹H NMR(DMSO-d₆, 300 MHz) δ8.25(br s,impurities, TsOH), 7.94–7.88(m, 2H), 7.81–7.74(q, J=6.2 Hz, 1H),7.71–7.62(m, 2H), 7.49(d, J=8.1 Hz, 1H), 7.12(d, J=8.1 Hz, 1H), 5.39(brs, 2H), 4.52–4.44(m, 1H), 4.41–4.33(m, 1H), 3.90–3.82(m, 1H), 3.78(t,J=5.5 Hz, 2H), 3.67(t, J=6.2 Hz, 1H), 3.44(t, J=6.6 Hz, 1H), 2.29(s, 3H+impurities, TsOH), 1.20(d, J=7.3 Hz, 3H) ¹³C NMR (DMSO-d₆, 75 MHz)δ169.5, 145.5, 139.3, 137.7, 134.1, 133.6, 129.5, 129.3, 128.0, 127.7,127.6, 125.5, 58.9, 57.5, 54.9, 53.6, 47.7, 20.7, 15.2: MS(ESI) m/z258.1 (100%, M—TsOH—H); IR KBr) ν_(max) 3424(br), 2927(br), 1745(m),1309(m), 1224(m), 1195(m), 1147(s), 1124(m), 1087(m), 1007(m) cm⁻¹;Anal. Calcd for C₁₈H₂₅NO₄S: C, 50.10; H, 5.84; N, 3.25; S, 14.86. Found:C, 48.49; H. 5.31; N, 2.56; S, 14.72.

To a slurry of benzyl N-acetyl-4,6-benzylidine muramic acid (20.0 g,42.5 mmol) in CH₂Cl₂ (300 mL) at 0° C. is added N-methylmorpholine (NM)(4.67 mL, 42.5 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (8.94 g,51.0 mmol). After stirring for 45 min at 0° C., CH₂Cl₂ (300 mL) followedby NMM (9.34 mL, 83.0 mmol) and L-alanine(phenylsulfonylethyl ester,tosylate salt) (15.4 g, 51.0 mmol) (i.e., compound i) are added to theabove reaction mixture. The resulting solution is slowly warmed to roomtemperature and stirred for 3 days. The reaction mixture is thenfiltered. The filtrate is washed first with 1N HCl then with brine, anddried (MgSO₄). The filtrate is then concentrated under reduced pressure,evaporated with toluene(×2), and vacuum dried overnight to afford theproduct, compound ii, (23.5 g, 95%) as a white solid.

Analytical (compound ii): ¹H MM (CDCl₃, 400 MHz) δ7.44(d, J=3.0 Hz, 2H),7.35(m, 8H), 6.95(d, J=6 Hz, 1H), 6.15(d, J=6.0 Hz, 1H), 5.85(m, 1H),5.47(s, 1H), 5.21(dd, J=3.0, 12.0 Hz, 1H), 5.30(dd, J=3.0, 15.0 Hz, 1H),4.90(d, J=3.0 Hz, 1H), 4.72(d, J=12.0 Hz, 1H), 4.60(m, 2H), 4.42(m, 2H),4.30–4.20(m, 2H), 4.15(q, J=3.0 Hz, 1H), 4.00(q, J=3.0 Hz, 1H), 3.82(m,1H), 3.75(d, J=9.0 Hz, 1H), 3.65(m, 1H), 1.93(s, 3H), 1.43(d, J=3.0, 9.0Hz, 3H), 1.38(d, J=3.0, 9.0 Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ173.2,172.2, 170.6, 131.6, 129.0, 128.9, 128.4, 128.3, 125.9, 101.4, 97.5,81.7, 78.3, 76.6, 75.6, 75.1, 70.1, 68.9, 65.8, 64.1, 63.2, 55.3, 53.2,48.1, 23.0, 17.4, 17.8. MS (PSI) m/z 583.2 (86%, M+H), 581.3(100%, M−H);IR ν_(max) (CHCl₃) 3010(m), 1740(m), 1681(s), 1616(m), 1569(s), 1523(m),1470(m), 1377(s), 1333(m), 1119(m), 1090(m) cm⁻¹; Anal. Calcd forC₃₁H₃₈N₂O₉: C, 63.90; H, 6.57; N, 4.81. Found: C, 63.78; H. 6.55; N,4.89.

Triethylsilane (16.4 mL, 103 mmol) is added to a solution of compound ii(12.0 g, 20.6 mmol) in CH₂Cl₂ (150 mL) at 0° C., followed by dropwiseaddition of TFA (8.1 mL, 103 mmol). The mixture is allowed to stir for 5h, after which an additional 3 equivalents of TFA (5.0 mL) is addeddropwise, and stirred at 0° C. overnight. Upon completion of thereaction, as evidenced by TLC (EtOAc), the reaction mixture is dilutedwith CH₂Cl₂, then NaHCO₃ is added slowly to neutralize the TFA. Theaqueous layer is extracted with CH₂Cl₂. The organic layer is washed withbrine (×2), then dried (MgSO₄), and concentrated in vacuo. Purificationby prep-LC (eluting with 70:30 EtOAc:hexane to EtOAc), followed byrecrystallization from CH₂Cl₂ and isopropyl ether gives the product,compound iii, (7.4 g, 61%) as a white solid.

Analytical (compound iii): ¹H NMR (CDCl₃, 300 MHz) δ7.38–7.26(m, 10H),6.99 d, i=7.3 Hz, 1H), 6.16(d, J=8.8 Hz, 1H), 5.94–5.81(m, 1H), 5.30(dd,J=1.1, 17.2 Hz, 1H), 5.22(dd, J=1.1, 10.6 Hz, 1H), 4.92(d, J=3.7 Hz,2H), 4.68(t, J=11.7 Hz, 1H), 4.59(d, J=7.7 Hz, 4H), 4.49(q, J =6.2 Hz,1H), 4.46(dd, J=2.2, 11.7 Hz, 2H), 4.21(dq, J=3.7, 9.9 Hz, 1H), 4.17(q,J=7.0 Hz, 1H), 3.83–3.75(m, 1H), 3.71(t, J=5.1 Hz, 1H), 3.68–3.65(m,1H), 3.54(t, J=10.2 Hz, 1H), 1.89(s, 3H), 1.44(d, J=7.0 Hz, 3H), 1.40(d,J=7.0 Hz, 3H); ¹³C NMR(CDCl₃, 75 MHz) δ173.0, 172.3, 170.3, 167.7,137.8, 137.1, 131.7, 128.6, 128.5, 128.1, 127.8, 127.7, 118.5, 97.1,80.5, 77.7, 73.7, 71.6, 70.5, 70.2, 69.8, 65.8, 55.1, 52.5, 48.0, 24.5,23.3, 19.2, 17.7; MS(ESI) m/z 585.2 (100%, M+H), 583.2 (100%, M−H); IRν_(max)(CHCl₃) 3433(m), 3010(m), 1741(m), 1677(s), 1522(m), 1454(m),1124(m), 1058(m) cm⁻¹; Anal. Calcd for C₃H₄₀N₂O₉: C, 63.68; H, 6.90; N,4.79; Found: C, 63.67; H, 6.58; N, 4.83.

II. Glycosidation

Compound iv is prepared using the procedures described in Imoto, M.,Bull. Chem. Soc. Jpn., 60, 2205 (1987).

To a solution of compound iii (4.59 g, 6.43 mmol) in CH₂Cl₂ (30 mL) areadded 4Å molecular sieves (10 g) and silver triflate (5.12 g, 20.0mmol). To this mixture is added a solution of freshly prepared compoundiv (10.8 g, 20.0 mmol) in CH₂Cl₂ (9.5 mL) in four portions over a 1 hperiod. Each of the starting materials is dried prior to use, and thereaction is performed under controlled anhydrous conditions. Afterstirring at room temperature for 24 h, the reaction mixture is filteredthrough Celite and washed with CH₂Cl₂. The organic layer is washed withNaHCO₃, brine, dried (Na₂SO₄), and concentrated in vacuo. Purificationby column chromatography on silica (Flash Elute system) utilizing asolvent gradient of 50% hexane in EtOAc, 15% hexane in EtOAc, EtOAc, and5% MeOH in EtOAc yields the product, compound v, (5.73 g, 76%) as awhite solid, along with unreacted starting material, compound iii, (630mg, 14%).

Analytical (compound v): ¹H NMR(CDCl₃, 300 MHz) δ7.91(d, J=7.0 Hz, 2H),7.66(t, J=7.3 Hz, 1H), 7.58–7.50(m, 4H), 7.45(t, J=7.3 Hz, 2H),7.33–7.26(m, 6H), 6.83(d, J=7.3 Hz, 1H), 6.52(d, J=7.0 Hz, 1H), 5.09(d,J =2.9 Hz, 1H), 4.97(t, J=9.5 Hz, 1H), 4.87(d, J=12.1 Hz, 1H),4.79–4.73(m, 2H), 4.60(dd, J=7.3, 12.1 Hz, 2H), 4.53–4.29(m, 5H),4.26–4.04(m, 7H), 4.00–3.88(m, 2H), 3.70–3.50(m, 4H), 3.42(t, J=10.6 Hz,4H), 2.03(s, 3H), 1.98(s, 6H), 1.89(s, 3H), 1.34(d, J=6.6 Hz, 3H),1.24(d, J=7.3 Hz, 3H); ¹³C NMR(CDCl₃, 75 MHz) δ173.4, 171.8, 170.6,170.3, 169.4, 154.1, 137.3, 134.0, 129.4, 129.1, 128.5, 128.1, 100.0,97.1, 96.9, 77.4, 77.0, 76.6, 75.7, 74.5, 73.8, 72.2, 71.2, 70.4, 70.3,68.3, 67.2, 61.5, 58.1, 26.2, 54.9, 53.6, 47.7, 23.2, 20.6, 18.3, 17.5;MS (FAB) m/z 1176.3 (73%, M+H), (ESI) m/z 1174.5 (62%, M−H) IR (KBr)ν_(max) 3385(br), 3067(w), 2939(w), 1753(s), 1669 m), 1537(m), 1233(s),1145(m), 1045(s) cm⁻¹; UV-vis (95% EtOH) λ_(max) 264 (1223.11)nm; Anal.Calcd for C₅₁H₆₄Cl₃N₃O₂₀S: C, 52.02; H, 5.48; N, 3.57; S, 2.72; Cl,9.03. Found: C, 51.72; H, 5.40; N, 3.64; S, 2.72; Cl, 9.07.

III. Protective Group Interchange

To a solution of compound v (1.9 g, 1.57 mmol) in Ac₂O:AcOH (2:1, 11 mL)is added a solution of ZnCl₂ (2.1 g, 15.7 mmol) in Ac₂O:ACOH (2:1, 5 mL)in one portion. Upon completion of the reaction (24 h) as judged by TLC(EtOAc), Troc is removed by adding Zn dust (4.1 g, 62.8 mmol) and amixture of THF:Ac₂O:AcOH (3:2:1, 25 mL) to the above reaction mixtureand stirring until no starting material is evidenced by TLC (EtOAc). Thereaction mixture is filtered through Celite, washed with EtOAc, and thenconcentrated under reduced pressure. The residue is repeatedlyevaporated with toluene to remove any remaining Ac₂O and AcOH, and thendiluted with EtOAc. The organic layer is washed with NaHCO₃ (×2), H₂O(×2), and brine. The organic layer is then dried (Na₂SO₄) andconcentrated in vacuo. Purification via column chromatography on silica(Flash Elute system) eluting with 2% MeOH in EtOAc affords the product,compound vi,(1.0 g, 67%) as a white solid.

Analytical (compound vi): ¹H NMR (CDCl₃, 300 MHz) δ7.89(d, J=7.0 Hz,2H), 7.66(t, J=7.3 Hz, 1H), 7.56(t, J=7.7 Hz, 2H), 7.34–7.23(m, 6H),7.16(d, J=7.7 Hz, 1H), 6.88(d, J=7.0 Hz, 1H), 6.12(d, J=9.5 Hz, 1H),5.12–5.07(m, 3H), 4.56 (dd, J=12.1, 40.0 Hz, 2H), 4.45(d, J=9.0 Hz, 1H),4.39(d, J=8.4 Hz, 1H), 4.35–4.23(m, 4H), 4.17(d, J=12.0 Hz, 2H),4.09–3.95(m, 3H), 3.78(d, J=5.5 Hz, 2H), 3.60–3.48(m, 3H), 3.41–3.30(m,2H), 2.12(s, 3H), 2.01(s, 3H), 2.00(s, 3H), l.99(s, 3H), 1.94(s, 3H),1.92(s, 3H),1.38 (d, J=6.6 Hz, 3H), 1.28(d, J=7.3 Hz, 3H); ¹³CNMR(CDCl₃, 75 MHz) δ173.8, 171.9, 171.2, 170.9, 170.8, 170.6, 169.3,139.2, 137.3, 134.1, 129.4, 128.9, 128.5, 128.1, 128.0, 127.8, 100.2,96.9, 77.1, 76.6, 75.9, 75.6, 72.5, 71.8, 70.2, 69.5, 68.2, 62.3, 61.6,58.0, 54.9, 54.6, 53.6, 47.8, 23.2, 23.1, 20.9, 20.6, 18.4, 17.3; MS(ESI) m/z 994.7 (100%, M−H); IR (KBr) ν_(max) 3384(br), 3301(br),3068(w), 2939(w), 1748(s), 1670(s), 1540(m), 1372(m), 1236(s), 1144(m),1041(s) cm⁻¹; Anal. Calcd for C₄₅H₆₁N₃O₂₀S: C, 54.26; H, 6.17; N, 4.22;S, 3.22. Found: C, 53.96; H, 5.78; N, 4.17; S, 3.09.

Scheme III and Example III, both shown below, illustrate the synthesisof GMDP from compound vi.

Example 2

I. Preparation of Compound vii

The phenylsulfonyl ester, compound vi, (549 mg, 0.52 mMol) is dissolvedin THF (15 mL). After stirring commences, DBU (90 μL, 0.6 mMol) is addeddropwise as a THF solution (5 mL). After 1.5 h, TLC analysis (10% MeOHin chloroform) indicates complete conversion to a new product at lowerR_(f). The reaction mixture is partitioned between EtOAc and 1 N HCl.The organic phase is layered with water and, with vigorous stirring, thepH is adjusted to 8.8 with 2 N NaOH (meter). In a similar manner, thebasic aqueous phase is layered with chloroform, the system is stirredvigorously while the pH is adjusted to 1.5 with concentrated HCl. Theacidic aqueous phase is extracted again with chloroform. The combinedchloroform solutions are dried (MgSO₄) and concentrated to give thedesired disaccharide acid, compound vii, as a colorless solid (412 mg,96%). ESI-MS (negative ion)=824.8.

II. Preparation of Compound ix

The disaccharide acid, compound vii, (1.02 g, 1.24 mMol) is slurried inacetonitrile. N-hydroxysuccinimide (156 mg, 1.36 mMol) and EDCI (261 mg,1.36 mMol) are then added. The system becomes homogeneous immediately.After 4 h, TLC analysis (10% MeOH in chloroform) indicates completeformation of the NHS active ester intermediate, i.e., compound viii. Asolution of γ-Obu^(t)-iso-Gln (275 mg, 1.36 mMol) in2:1=acetonitirile:DMF (5 mL) is added dropwise, followed immediately bydiisopropylethyl amine (237 μL, 1.36 mMol). After 3.5 h, TLC analysis(10% MeOH in chloroform) indicates complete conversion of the activeester to the desired glycodipeptide at R_(f)=0.32. A small amount of avery slightly higher R_(f) product, possibly the diastereomer at Ala, isalso observed. The reaction mixture is partitioned between EtOAc and NHCl. The organic phase is dried (MgSO4) and concentrated to a solid. Thecrude product is adsorbed on silica gel (10 g), and chromatographed oversilica gel (10 g) using an elution gradient of chloroform to 10% MeOH inchloroform. The product thus obtained, tetraacetyl glycodipeptide(compound ix), (colorless solid, 1.10 g, 88%) is diastereomericallypure. ESI-MS (positive ion)=1010.4, 1032.4

III. Preparation of GMPD

The tetraacetyl glycodipeptide (compound ix) (1.02 g, 1.0 mMol) isdissolved in dry MeOH (25 mL). A solution of 0.5 M NaOMe in MeOH (2.0mL, 1 mMol) is added with stirring.

The reaction mixture is stirred at room temperature until ESI-MSanalysis indicates that all four acetyl groups have been removed,thereby forming the tetrahydroxy plycopeptide (compound x) (positiveion, M+H=843). Dowex resin 50WX8-400 is added portionwise with stirringuntil the apparent pH (paper) reaches 4–5. The resin is removed byfiltration and the solution concentrated to a thick oil. The oil istaken up in 0.25 M HCl in ethanol and stirred at room temperature for 3hr. Pd/C (0.5 g) is added to the reaction mixture, and the system isbrought under a hydrogen atmosphere. After 2.5 h, the catalyst isremoved by filtration and the filtrate concentrated in vacuo. Theconcentrate is lyophilized twice from saturated aqueous NH₄HCO₃ toafford GMDP as an off-white solid (598 mg, 86%). ESI-MS (positive ion,M+14=709.4; negative ion, M−H=693.9).

1. A process for preparing a protected glycopeptide of formula 1

comprising: coupling a muramylamide compound of formula 2

with a glucopyranosyl compound of formula 3

to form said glycopeptide of formula 1, wherein: A is Br or Cl; Pg⁰ isan acyl hydroxy-protecting group; Pg¹ is a hydroxy-protecting groupwhich is not electron withdrawing; Pg² is an amine-protecting groupwhich does not lead to oxazoline formation; Pg⁵ is a hydroxy-protectinggroup; Pg⁰, Pg¹, Pg² and Pg⁵ are orthogonal protecting groups; and Y isa residue of an amino acid or peptide of 2 to 5 amino acid residues,wherein: Y forms an amide linkage with the attached carbonyl; and Ycomprises a protected terminal carboxy group.
 2. The process of claim 1,wherein said reacting is carried out under extreme anhydrous conditions.3. The process of claim 1, wherein Pg¹ is benzyl.
 4. The process ofclaim 3, wherein said muramylamide of formula 2 is prepared byreductively opening the 1,3-dioxane ring of a compound of formula 6

wherein: Pg⁵ is a hydroxy-protecting group which does not lead tooxazoline formation; and Y is a residue of an amino acid or peptide of 2to 5 amino acid residues, wherein: Y forms an amide linkage with theattached carbonyl; and Y comprises a protected terminal carboxy group.5. The process of claim 1, further comprising: exchanging said Pg¹ groupof said compound of formula 1 with a Pg³ group to form a compound offormula 10

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg² is anamine-protecting group which does not lead to oxazoline formation; Pg³is acyl hydroxy-protecting group; Pg⁵ is a hydroxy-protecting group;Pg³, Pg², and P⁵ are mutually orthogonal protecting groups; and Y is aresidue of an amino acid or peptide of 2 to 5 amino acid residues,wherein: Y forms an amide linkage with the attached carbonyl; and Ycomprises a protected terminal carboxy group.
 6. The process of claim 5,wherein Pg¹ is benzyl.
 7. The process of claim 5, wherein Pg³ is acetyl.8. The process of claim 5, further comprising: exchanging said Pg² groupof said compound of formula 10 acetyl group to form a compound offormula 9

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ is an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting group; Pg⁰ and Pg³are orthogonal to Pg⁵; and Y is a residue of an amino acid or peptide of2 to 5 amino acid residues, wherein: Y forms an amide linkage with theattached carbonyl; and Y comprises a protected terminal carboxy group.9. The process of claim 8, wherein Pg² is 2,2,2-trichloroethoxycarbonyl.10. The process of claim 9, wherein Pg¹ is benzyl and Pg³ is acetyl. 11.The process of claim 10, wherein said exchanging of said Pg¹ and Pg²groups comprises: (a) dissolving said compound of formula 1 in aceticanhydride and acetic acid; (b) adding anhydrous zinc chloride toexchange said Pg¹ group with an acetyl group; and (c) adding zinc dustto exchange said Pg² group with said acetyl group.
 12. The process ofclaim 8, further comprising: deprotecting said terminal carboxy group ofsaid Y group of said compound of formula 9 to form a compound of formula8

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ is an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting group; Pg⁰ and Pg³are orthogonal to Pg⁵; and Y′ is a residue of an amino acid or peptideof 2 to 5 amino acid residues wherein Y forms an amide linkage with theattached carbonyl.
 13. The process of claim 12, further comprising:saponifying said Pg⁰ and Pg³ groups of said compound of formula 9 toform a compound of formula 7

wherein: Pg⁵ is a hydroxy-protecting group; and Y′ is a residue of anamino acid or peptide of 2 to 5 amino acid residues, where Y′ forms anamide linkage with the attached carbonyl.
 14. The process of claim 13,further comprising: removing said Pg⁵ group of said compound of formula7 to form a compound of formula I

wherein: Y′ is a residue of an amino acid or peptide of 2 to 5 aminoacid residues; and Y′ forms an amide linkage with the attached carbonyl.15. The process of claim 14, wherein Pg⁵ is benzyl.
 16. The process ofclaim 1, wherein said glycopeptide is of formula 1a

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg¹ is ahydroxy-protecting group which is not electron withdrawing; Pg² is anamine-protecting group which does not lead to oxazoline formation; Pg⁵is a hydroxy-protecting group; Pg⁰, Pg¹, Pg², and Pg⁵ are mutuallyorthogonal protecting groups; and X is a residue of an amino acid orpeptide of 2 to 4 amino acid residues, wherein: X forms an amide linkagewith the attached carbonyl; and X comprises a protected terminal carboxygroup.
 17. The process of claim 16, further comprising: exchanging saidPg¹ group of said compound of formula 1a with a Pg³ group to form acompound of formula 19

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg² is anamine-protecting group which does not lead to oxazoline formation; Pg³is an acyl hydroxy-protecting group; Pg⁵ is a hydroxy-protecting group;Pg^(3, Pg) ² and Pg⁵ are mutually orthogonal protecting groups; and X isa residue of an amino acid or peptide of 2 to 4 amino acid residues,wherein: X forms an amide linkage with the attached carbonyl; and Xcomprises a protected terminal carboxy group.
 18. The process of claim17, wherein Pg¹ is benzyl.
 19. The process of claim 17, wherein Pg³ isacetyl.
 20. The process of claim 17, further comprising: exchanging saidPg² group of said compound of formula 19 with an acetyl group to form acompound of formula 18

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ is an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting group; Pg⁰ and Pg³are orthogonal to Pg⁵; and X is a residue of an amino acid or peptide of2 to 4 amino acid residues, wherein: X forms an amide linkage with theattached carbonyl; and X comprises a protected terminal carboxy group.21. The process of claim 20, wherein Pg² is2,2,2-trichloroethoxycarbonyl.
 22. The process of claim 21, wherein Pg¹is benzyl and Pg³ is acetyl.
 23. The process of claim 22, wherein saidexchanging of said Pg¹ and Pg² groups comprises: (a) dissolving saidcompound of formula 1a in acetic anhydride and acetic acid; (b) addinganhydrous zinc chloride to exchange said Pg¹ group with an acetyl group;and (c) adding zinc dust to exchange said Pg² group with said acetylgroup.
 24. The process of claim 20 further comprising: deprotecting saidterminal carhoxy group of X group of said compound of formula 18 to forma compound of formula 17

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ in an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting group; Pg⁰ and Pg³are orthogonal to Pg⁵; and X′ is a residue of an amino acid or peptideof 2 to 4 amino acid residues, where X′ forms an amide linkage with theattached carbonyl.
 25. The process of claim 24, further comprising:reacting said compound of formula 17 with a compound of formula LOH toform an activated ester of formula 15

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ is an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting group; Pg⁰ and Pg³are orthogonal to Pg⁵; X″C(O)OL is the activated ester of X′; —OL is aleaving group susceptible to displacement by an amine nucleophile; andX′ is a residue of an amino acid or peptide of 2 to 4 amino acidresidues, where X′ forms an amide linkage with the attached carbonyl.26. The process of claim 25, wherein LOH is N-hydroxysuccinimide. 27.The process of claim 25, further comprising: coupling said compound offormula 15 with a compound of formula WH to form a compound of formula13

wherein: Pg⁰ is an acyl hydroxy-protecting group; Pg³ is an acylhydroxy-protecting group; Pg⁵ is a hydroxy-protecting; Pg⁰ and Pg³ areortogonsi to Pg⁵; X′ is a residue of an amino acid or peptide, where X′forms an amide linkage with the attached carbonyl; W is a residue of anamino acid or peptide, where W comprises a protected terminal carboxygroup; and W and X′ together consist of 2 to 5 amino acid residues. 28.The process of claim 27, further comprising: saponifying said Pg⁰ andPg³ groups of said compound of formula 13 to form a compound of formula12

wherein: Pg⁵ is a hydroxy-protecting; X′ is a residue of an amino acidor peptide, where X′ forms an amide linkage with the attached carbonyl;W is a residue of an amino acid or peptide, where W comprises aprotected terminal carboxy group; and X′ and W together consist of 2 to5 amino avid residues.
 29. The process of claim 28, wherein Pg⁰ isacetyl.
 30. The process of claim 28, wherein Pg³ is acetyl.
 31. Theprocess of claim 28, further comprising: removing said Pg⁵ group of saidcompound of formula 12 to form a compound of formula 11

wherein: X′ is a residue of an amino acid or peptide, where X′ forms anamide linkage with the attached carboxyl; W is a residue of an aminoacid or peptide, where W comprises a protected terminal carboxy group;and X′ and W together consist of 2 to 5 amino acid residues.
 32. Theprocess of claim 31, wherein Pg⁵ is benzyl.
 33. The process of claim 31,further comprising: deprotecting said W group of said compound offormula 11 to form a compound of fonnula I

wherein: —Y′ is —X′—W′; X′ is a residue of an amino acid or peptide,where X′ forms an amide linkage with the attached carbonyl; W′ is aresidue of an amino acid or peptide and X′ and W′ together consist of 2to 5 amino acid residues.
 34. The process of claim 1, wherein A is Br.35. The process of claim 1, wherein Pg² is a carbamate or imideamine-protecting group.
 36. The process of claim 35, wherein Pg² is acarbamate or imide amine-protecting group.
 37. The process of claim 36,wherein Pg² is 2,2,2-trichloroethoxycarbonyl.
 38. The process of claim1, wherein Pg¹ is a benzyl, allyl or silyl hydroxy-protecting group. 39.The process of claim 38, wherein Pg¹ is benzyl.
 40. The process of claim1, wherein Pg⁰ is acetyl.
 41. The process of claim 1, wherein Pg⁵ is abenzyl, allyl or n-pentenyl hydroxy-protecting group.
 42. The process ofclaim 41, wherein Pg⁵ is benzyl.
 43. The process of claim 1, wherein Yis a linear peptide.
 44. The process of claim 16, wherein X is a linearpeptide.
 45. The process of claim 27, wherein: X′ is a peptide of 2–4amino acid residues; and W is a peptide of 2–4 amino acid residues,provided that X′ and W together consist of 2 to 5 amino acid residues.46. The process of claim 27, wherein —X′—W is a linear peptide.